All organisms require metals as co-factors, and these essential metals must be obtained from external sources. The outer membrane of Gram-negative bacteria contains transporters specific for different organometallic compounds;in pathogenic bacteria, these transporters are implicated as virulence factors. As the outer membrane does not maintain an electrochemical gradient (pmf), energy-dependent steps in transport across the outer membrane are mediated through a coupling protein, TonB, that links the pmf-utilizing ExbB/ExbD/TonB motor of the inner membrane to the outer-membrane transporter. To date, seven TonB-dependent transporter structures have been determined by x-ray crystallography, including our structure of the cobalamin transporter BtuB. Their common architecture consists of a twenty-two stranded ?-barrel situated in the membrane, long extracellular loops, short periplasmic turns and a distinctive luminal domain. This luminal domain, comprised of the amino-terminal portion of the transporter, forms a globular-like domain that occludes the barrel. These structures are mechanistically provocative: what conformational changes does the luminal domain undergo to permit substrate permeation through the transporter? how does TonB couple to the transporter? what are the mechanistic and energetic features of the active transport cycle? what are the characteristics of the ExbB/ExbD/TonB motor? We will apply structural biology results to other experimental (and computational) methods directed to the central mechanistic questions of TonB-dependent outer membrane active transport. Specifically, we will: (1) determine crystal structures of mutant BtuB, BtuB:cobalamin and BtuB:TonB:cobalamin complexes;(2) characterize periplasmic exposure of individual residues of the luminal domain during the transport cycle, and combine these data with computer simulations for design of additional experiments;(3) determine structures of individual proteins and sub- complexes of proteins of the ExbB/ExbD/TonB motor;and (4) isolate and characterize the ExbB/ExbD/TonB motor for use in subsequent functional and structural studies. A long-term goal of this research is to develop novel antibacterial compounds that target specifically TonB-dependent transport. PUBLIC HEALTH RELEVANCE Gram-negative bacteria such as Escherichia coli require micronutrients (such as vitamin B12 and iron) for survival. They take up these scarce materials by way of specialized transport systems that use the energy of the bacterial cell to bring these materials into the cell. This research is directed towards understanding how a portion of this transport pathway works, and a long-range goal of this research is to develop antibacterial drugs targeted to this transport pathway.